The transparent cornea is made up of an anterior stratified epithelium, a collagenous stromal matrix containing fibroblasts called keratocytes, Descemet's membrane and a single-layered endothelium. We have put forth the refracton hypothesis attempting to relate cornea to lens. The cornerstone of this hypothesis is that the exceptionally abundant intracellular proteins in the cornea share properties with the multifunctional crystallins of the lens. Our current corneal research addresses the molecular basis for corneal-specific gene expression, the roles of the abundant, intracellular corneal proteins, the global patterns of corneal gene expression, and stratification of the corneal epithelium. For corneal-specific gene expression, we are continuing to investigate the mouse aldehyde dehydrogenase 3 (ALDH3a1) and zebrafish gelsolin promoter in both transfection and transgenic mouse experiments; we have also finalized a study on rabbit ALDH1a1 gene expression in the cornea. In FY2002 we showed that gelsolin, an actin binding protein, comprises approximately half of the water-soluble protein of the zebrafish corneal epithelium, making it a putative corneal crystallin in this species, and then in FY2003 we reported that gelsolin is expressed at the blastula stage in zebrafish embryos and in the notocord before it becomes concentrated in the eye. Moreover, microinjection of human gelsolin, zebrafish gelsolin mRNA, and gelsolin-specific morpholino oligonucleotides established that zebrafish gelsolin is required for dorsalization during embryogenesis, consistent with it having a signal transduction role during development. Indeed, injection of gelsolin mRNA or human gelsolin protein dorsalized the developing embryo, often resulting in partial axis duplication. Thus, gelsolin, which is specialized for corneal expression in adult zebrafish, also modulates embryonic dorsal/ventral pattern formation in this species. Current experiments in FY2004 provided evidence that the signal transduction role of the corneal-enriched gelsolin during development involves binding of BMP4, inhibiting its known ventralizing potential. In FY2003 we initiated a cloning project for a second gelsolin gene in zebrafish. In FY2004 we established that the second gelsolin gene is expressed in many tissues and is not overexpressed in the cornea. Of particular interest, it appears that this ubiquitously expressed gelsolin gene does not influence dorsal/ventral patterning in zebrafish as does the gelsolin that is enriched in the cornea. The two gelsolins share approximately 60% amino acid sequence identity. We have recently provided evidence that the ubiquitously expressed gelsolin affects macrophage motility during development. Thus, the two gelsolin genes in the zebrafish have different expression patterns and functions. The molecular basis for the corneal-preferred activity of the gelsolin promoter is under investigation. We are continuing to investigate the molecular basis for the high corneal expression of mouse ALDH3a1 in corneal epithelial cells of transgenic mice, which also represents approximately 50% of the water-soluble protein in this transparent tissue. We demonstrated in FY2003 that the mouse ALDH3a1 promoter is activated by Pax6 in co-transfection experiments, a result consistent with our earlier demonstration that this gene is down-regulated in the Small eye mice that have reduced Pax6 expression in the cornea. In FY2004 we provided evidence that a GC-rich sequence in the first intron of the ALDH3a1 gene acts as a strong suppressor of promoter activity, suggesting new avenues of investigation for understanding the tissue-regulated expression of this gene. In FY2002 we began to explore global gene expression in 6-week-old and 9-day-old mouse corneas by making and analyzing SAGE (serial analysis of gene expression) libraries. 60,000 tags, each representing the expression of a cDNA, were sequennced from each stage. In short, the paattern of genes expressed is characteristic of the tissue and is in good agreement with what is known about the expression of corneal proteins. Numerous genes expressed in these libraries are not represented in published SAGE libraries from other tissues (e.g. retina, lymphocytes, etc.). Many genes were identified that either increase or decrease in expression between these two stages of corneal development. In brief, our SAGE data demonstrate dynamic changes in gene expression after eye opening, and provide new probes for exploring corneal epithelial cell stratification, development and function, and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. In FY2004 we have extended our SAGE experiments to the cornea of newborn mice and these data are presently being analyzed. We are also completing an analysis of earlier SAGE data on global gene expression in the limbal stem cells and central epithelial cells of the adult rat. We anticipate that together these fundamental data on corneal gene expression will have many applications in the future to biomedical problems of the cornea.